1. Field of the Invention
The present invention relates to a biologically active substance consisting of specific compounds. Further, the present invention relates to a melanin production inhibitor, cell growth activator, anti-bacterial agent, etc. containing the above-mentioned compounds. Further, the present invention relates to an agent for external application onto the skin, oral cavity compositions, bath additives, etc.
2. Prior Art
Compounds with a wide variety of biological activities have been developed heretofore. In particular there are a large number of reports on compounds derived from natural materials especially from the safety point of view.
As typical compounds having biological activity, those having an inhibitory effect on production of melanin are described hereinafter.
As age advances, stains, freckles or deposition of pigment after sunburn tends to be formed, increased or settled into the skin. This is problematic particularly for the middle-aged and the elderly.
Although there are still unrevealed aspects of the mechanism of generation of such acquired pigment (melanin) deposition, it is believed that melanin pigment formation is caused by hormone abnormality and external stimuli such as ultra violet rays from sunlight, oxygen and chemical substances etc., and abnormally deposited in the skin. The development of compounds preventing the formation and deposition of this melanin is highly desired and many compounds have been developed therefor. These compounds include ascorbic acid and derivatives thereof, a placenta extract, hydroquinone, kojic acid, arbutin and ellagic acid, and further there are a large number of reports on melanin production-inhibiting components extracted from plants, and these include a chamomile extract (Matricaria chamomilla L. (Composite)) disclosed in Japanese Patent Application Laid-Open No. 8-92056, a golden flower root extract (Scutellaria baicalensis G. (Labiatae)) disclosed in Japanese Patent Application Laid-Open No. 8-104616, a cumin seed (Cuminum cyminum L. (Umbelliferae)) disclosed in Japanese Patent Application Laid-Open NO. 8-119848 and a wolo extract (Borassus flabellifera (Palmac)) disclosed in Japanese Patent Application Laid-Open No. 10-29928.
Further, the present inventors also found that a fraction obtained by purifying solvent extracts from various plants by silica gel chromatography strongly inhibits the production of melanin in B16 melanoma cells, and filed for a patent (Japanese Patent Application No. 9-254025, Aug. 15, 1997). As a substance having a labdan structure, manool, an extract of Dacrydium biforme is reported to have an inhibitory effect on production of melanin (Japanese Patent Application Laid-Open No. 6-72855) Further the inhibitory effect of derivatives thereof on melanin production is also reported (Japanese Patent Application Laid-Open Nos. 7-25754, 7-69858, 7-206625 etc.).
However, the majority of these conventional melanin production inhibitors are inadequate with respect to stability, effects, adverse side effects, etc., so a new melanin inhibitor has been desired.
Cell growth activators are now described hereinafter. In aged skin, the activity of skin cells is weakened so that wrinkles and flabby skin are formed. Recently, there are many studies in which skin cells themselves are activated and the function of the skin itself is thus activated to improve skin conditions, and attention has been paid to the development of cell growth activators for activating weakened cells as well as to the incorporation of such cell activators into an agent for external application onto the skin. Conventional materials used for imparting cell-activating activity include alpha-hydroxy acids such as glycolic acids, single-component materials such as hormones, vitamins, photosensitive elements, allantoin, etc., and extracted components including animal and plant extracts such as placenta extract, lactobacillus extract, shikon extracts, aloe extract, carrot extract, etc. Further, the present inventors also found that there is strong cell-activating activity in distillated residues of solvent extracts, etc. from various plants, and filed for a patent (Japanese Patent Application Laid-Open No. 8-284572, Oct. 8, 1996). Further, Labdanum furanoid diterpenoids (WO 97/45099) are reported as cell differentiation-inducing materials having a labdan structure. However, the majority of conventional materials and extracts having cell-activating activity are unsatisfactory with respect to their effects, so that they have to be applied in a large amount. Also their stability in storage is not satisfactory. Moreover, they may create safety problems because of their stimulating properties or similar nature.
Anti-bacterial agents are now described hereinafter. A large number of microorganisms are present on the skin and many of them are not problematic to a healthy skin, but under bad skin conditions or bad general conditions, these microorganisms invade hair follicles, sweat glands and damaged sites to act as causative agents for infections.
In addition, there are some microorganisms which cause body odors or dandruff or oxidize secreted lipids to exert adverse effects an causative factors for acne. To kill such microorganisms, many compounds have been used, but many of them are chemically synthesized products, so there has been a demand for highly safe anti-bacterial agents derived from natural sources.
The anti-bacterial activity of Cistus absolute as one of the extracts from Cistus ladaniferus L., Cistus creticus L., Cistus monoperiensis L., Cistus salvifolius, etc. has already been reported (Nippon Keshohin Gijyutusha Kaishi, 27, 227 (1993)), but its active ingredient is not referred to therein.
The object of the present invention is to provide compounds which have a wide variety of biological activities, are derived from natural sources, and are safe and user-friendly. In particular, the object of the present invention is to provide compounds having an inhibitory activity on production of melanin, a cell-activating activity and an anti-bacterial activity.
As a result of their eager study to solve this problem, the present inventors found that extracts with hot water, or extracts with ethanol, hexane, etc. of stems, branches, leaves, etc. of Cistus ladaniferus L., Cistus creticus L., Cistus monoperiensis L., Cistus salvifolius , etc. have a strong inhibitory activity on production of melanin, a cell-activating activity and an anti-bacterial activity, that these actions are based on labdanolic acid, and further that labd-7-en-15-oic acid, labd-8(17)-en-15-oic acid, and labd-8-en-15-oic acid obtained by molecular distillation of the above extracts, or of labdanolic acid have a strong inhibitory activity on production of melanin, a cell-activating activity and an anti-bacterial activity. Further, the present inventors found that salts thereof or methyl and ethyl ester derivatives thereof and reduced derivatives thereof have also the same activity, and as a result of additional examination, the present invention was completed at last. The biologically active substance of the present invention is one or more compounds represented by the following general formula (1): 
wherein R1 represents xe2x80x94CH2OH, xe2x80x94COOR6, or xe2x80x94COOX, where X is a group capable forming a salt and R6 represents hydrogen or a C1 to C3 lower alkyl group; R2 to R5 each represent hydrogen or a methyl group; and . . . A . . . represents xe2x95x90C(CH3)xe2x80x94, xe2x80x94C(CH3)xe2x95x90, xe2x80x94C(xe2x95x90CH2)xe2x80x94, xe2x80x94CH(CH3)xe2x80x94 or xe2x80x94C(OH)(CH3)xe2x80x94.
In the formula, X includes a group capable of forming a salt such as sodium, potassium, ammonium, etc., and R6 includes hydrogen, a methyl group, an ethyl group and a propyl group.
In the present invention, the biologically active substance refers to a substance having one or more activities selected from an inhibitory activity on melanin production, a cell-activating activity and an anti-bacterial activity.
The above compounds are those known in the art and their processes are also known. For example, labdanolic acid is a component in labdanum gum extracted from Cistus ladaniferus (J. Chem. Soc., 1956, 4259-4262), and labd-8(17)-en-15-oic acid (eperuic acid) and labd-8-en-15-oic acid are obtained by chemically treating labdanolic acid (J. Chem. Soc., 1956, 4262-4271). Further, it is reported that eperuic acid is a component in a resin derived from an Eperua falcata tree of the Leguminosae (J. Chem. Soc., 1955, 658-662), and labd-7-en-15-oic acid (cativic acid) is a component in a resin from Prioria copaifera G. tree of the Leguminosae (J. Am. Chem. Soc., Vol. 79, 1201-1205, 1957).
However, it was not known which biological activity these substances possess, neither was it known that they possess an inhibitory activity on production of melanin, a cell-activating activity and an anti-bacterial activity.
Although the plants used for preparing the compounds defined in the present invention are not particularly limited insofar as they are plant containing said compounds, it is particularly advantageous to employ Cistus ladaniferus L., Cistus creticus L., Cistus monoperiensis L., and Cistus salvifolius plants (Cistaceae family). These are used alone or in combinations thereof. The site of plant used is not particularly limited, and use is made of leaves, branches, stems, barks, etc. These may be used just after being harvested or after being dried.
Preferably, the method of extracting the desired compounds from said plants makes use of one or more solvents selected from the group consisting of water, lower alcohols, petroleum ether and hydrocarbons. The lower alcohols are those containing 1 to 4 carbon atoms, preferably methanol, ethanol, etc.
The petroleum ether used may be not only the one known in the art but also a commercial product.
The hydrocarbon solvents are aliphatic hydrocarbons, alicyclic hydrocarbons and aromatic hydrocarbons which are liquid at ordinary temperatures and, preferably, aliphatic hydrocarbons and aromatic hydrocarbons which are liquid at ordinary temperatures, among which hydrocarbons such as hexane and toluene are particularly preferable.
Although the operation of extraction differs depending on the plant and solvent used, usually divided pieces of the plant are immersed in the solvent, optionally under gentle stirring, at room temperature to a temperature of 50xc2x0 C.
Further, a soxhlet extractor known in the art may also be used.
The time required for extraction is usually 3 to 48 hours.
Alternatively, a method of steam distillation or boiling in hot water after leaves, branches or stems of the plant are disrupted may also be adopted in the present invention. In this case, gum which floats on water upon steam distillation or hot-water extraction is removed and then separated from insolubles by means of solvent extraction.
Further, commercially available products obtained from the above plants by any of the methods described above may be used.
The crude extract thus obtained contains 25 to 35% labdanolic acid. This crude extract itself may be used as a melanin production inhibitor, cell activator and anti-bacterial agent.
Hereinafter, a typical method of obtaining the acid or a mixture of the acids from the crude extract or from a commercially available extract is described, but the present invention is not limited to this example.
The above crude extract or a commercially available extract is subjected to molecular distillation under reduced pressure at 0.1 to 0.5 mm Hg whereby a fraction at 160 to 230xc2x0 C. and, preferably, 180 to 220xc2x0 C. is collected. This fraction contains a mixture of labd-7-en-15-oic acid, labd-8(17)-en-15-oic acid and labd-8-en-15-oic acid.
As the melanin production inhibitor, cell activator and anti-bacterial agent, this acid mixture may be used as such or if necessary as salts or methyl or ethyl ester derivatives thereof.
Then, the three acids are separated from this acid mixture.
Specifically, this acid mixture is dissolved in ethanol, then reacted to form ethyl ester derivatives in the presence of a catalytic amount of sulfuric acid, and subjected to silica gel chromatography on silica gel treated with silver nitrate. The column is washed with hexane and then the ethyl ester is eluted with 1% ethyl acetate-hexane. First, labd-8-en-15-oic acid ethyl ester is eluted and then labd-7-en-15-oic acid ethyl ester and labd-8(17)-en-15-oic acid ethyl ester are eluted in this order. The solvent is distilled off whereby purified products of the respective ethyl ester derivatives are obtained. Each of the ethyl ester derivatives thus obtained is hydrolyzed to give a free acid, and the free acid is further reacted with diazomethane to give its methyl ester derivative.
The resulting acids, methyl esters, ethyl esters or mixtures thereof are useful as melanin production inhibitors, cell activators, and anti-bacterial agents.
Further, these can be incorporated into an agent for external application onto the skin, a bath additive, an oral cavity composition, etc. to give a corresponding agent having an inhibitory activity on production of melanin, a cell-activating activity and an anti-bacterial activity. Further, the compound (1) of the present invention can be added to prepare an anti-aging agent and anti-wrinkle agent, etc.
The amount of said melanin production inhibitor, cell activator and anti-bacterial agent incorporated is 0.01 to 10% by weight, preferably 0.05 to 5% by weight, for the agent for external application onto the skin, 0.1 to 10% by weight, preferably 0.2 to 5% by weight, for the bath additives, 0.1 to 10% by weight, preferably 0.2 to 5% by weight, for an oral cavity composition, and 0.01 to 5% by weight, preferably 0.05 to 2% by weight, for an anti-bacterial agent.
The amount of the compound (1), if incorporated into face lotion, milky lotion, cream, etc., is usually 0.05 to 10% by weight, preferably 0.05 to 2% by weight.
The method of incorporation of the compound (1) into a melanin production inhibitor, a cell activator, an anti-bacterial agent, etc. is not particularly limited. For example, the compound(s) of the invention may be incorporated after being diluted with a usual organic solvent used in perfumes, that is, ethylene glycol, propylene glycol and lower alcohols which are used alone or as a mixture thereof, or after being diluted with a mixture of such solvent and a surface active agent, or after being mixed with conventional perfume materials. Alternatively, it may be incorporated as such in the absence of other materials.
Further, the melanin production inhibitor, cell activator, anti-bacterial agent, etc. of the present invention can contain not only the above essential ingredients but also other ingredients used in agents for external application onto the skin, such as usual cosmetics, quasi drug preparations, pharmaceutical preparations, etc. For example, it is possible to incorporate skin whitening agents, cell activators, humectants, antioxidants, oil components, surface active agents, thickeners, inorganic fillers, coloring agents, pH adjusters, preservatives, perfumes, UV absorbers, various skin nutrients, etc., depending on the object or on necessity.
Hereinafter, some of these ingredients are exemplified. As the skin whitening agents, mention can be made of arbutin, kojic acid, ellagic acid, ascorbic acid, etc. and various derivatives thereof, as well as extracts from various animals and plants such as placenta extract, etc. As the cell activators, mention is made of alpha-hydroxy acids such as glycolic acid, etc., hormones, vitamins, various animal and plant extracts. The humectant includes sorbitol, xylitol, glycerin, propylene glycol, sodium pyrrolidonecarboxylate, lactic acid, hyaluronic acid, collagen etc.; the antioxidants include vitamin E, butylhydroxytoluene, butylhydroxyanisol etc.; the oil components include vegetable fats and oils such as liquid paraffin, paraffin, olive oil, coconut oil, etc. and animal fats and oils such as tallow, porcine fat, mink oil, squalane, etc., and synthetic oils such as methyl polysiloxane, silicone oil, glyceryl triisopalmitate, etc.
The surface active agents include anionic surface active agents such as sodium lauryl sulfate, triethanolamine laurate, etc., cationic surface active agents such as cetyl trimethyl ammonium chloride, bonzalkonium chloride, etc., nonionic surface active agents such an glyceryl monoolearate, sorbitan monostearate, polyoxyethylene hydrogenated castor oil, and sucrose fatty acid ester; the thickeners include carboxymethylcellulose, hydroxyethylcellulose, carboxy-vinylpolymers, sodium alginate carrageenan, etc.; the inorganic fillers include talc, sericite, mica, kaolin, zinc white, titanium oxide, magnesium oxide, etc.; the pH adjusters include organic acids, such as citric acid, sodium citrate, etc. and salts thereof; the preservatives include urea, parabens such as methyl paraben, ethyl paraben, etc., sodium benzoate, ethyl alcohol, etc. Further, by adding various UV absorbers, it is also possible to improve the effect of preventing sunburns.